Construction of knockdown animal by transferring double-stranded rna expression vector

ABSTRACT

This invention relates to a target gene-knockdown animal and an animal model for disease in which mRNA of an endogenous target gene is disrupted via formation of double-stranded RNA for the target gene and a method for producing the animals. Also, this invention relates to a ds-RNA expression vector used for such method and an animal cell having such vector introduced therein.

TECHNICAL FIELD

The present invention relates to a target gene-knockdown animal, ananimal model for disease in which mRNA of an endogenous target gene isdisrupted via formation of double-stranded RNA (ds-RNA) for the targetgene and a method for producing such animals. Also, the presentinvention relates to a ds-RNA expression vector used for theaforementioned method and an animal cell into which such vector has beenintroduced.

BACKGROUND ART

Mice carrying specific gene mutations play key roles as murine modelsfor disease in fields of applied science as well as in fields of basicscience, such as embryology. Murine models for disease are very usefulfor the development of diagnostic techniques and therapeutic agents forvarious types of diseases. The method for producing mutant mice with theuse of ES cells, which is generally employed at present, was developedvia basic research conducted by Capecchi et al. (Capecchi, M R., Science(U.S.A.), 1989, vol. 244, pp. 1288-1292 (review)). In this technique,mutant mice are produced in the following procedures: 1) the genomicclone of the target gene is first separated, and a targeting vector inwhich the exon region essential for the functions has been substitutedwith a drug (e.g., neomycin)-resistant gene is constructed; 2) theresulting targeting vectors are introduced into ES cells byelectroporation or other methods, and recombinant cells in which thegenes of ES cells have been substituted with targeting vectors viahomologous recombination are separated; 3) the resulting recombinantcells are introduced into 8-cell-stage embryos to prepare chimeric mice;and 4) mice in which germ cells derived from the ES cells are formed areselected from the resulting chimeric mice, and homozygous mutant miceare obtained via mating. Since the methods of producing mutant mice thatare generally employed at present involve a large number of steps, itwould generally take for about 1 year and enormous labor to carry outsuch methods.

A phenomenon referred to as “RNA interference” has drawn attention inrecent years. This was originally found in, for example, nematodes (C.elegans), Drosophila, or plants. When double-stranded RNA (ds-RNA)corresponding to a specific mRNA sequence is introduced into a cell,mRNA transcribed in that cell is immediately disrupted (see Hannon, GJ., Nature, 2002, vol. 418, pp. 244-251 (review); JP Patent Publication(Kohyo) Nos. 2002-516062 A (WO 99/32619); 8-506734 A (1996); 2002-507416A (WO 99/49029); and 2003-516124 (WO 01/29058)). In recent years, suchRNA interference has been found to occur in cultured animal cells oreven in mice (see Elbashir, S M. et al., Nature, 2001, vol. 411, pp.494-498; McCaffrey, A P. et al., Nature, 2002, vol. 418, pp. 38-39; JPPatent Publication (Kohyo) Nos. 2002-502012A (WO 00/44895) and2003-514533 A (WO 01/36646)). A series of analyses that have beenconducted up to the present demonstrate that ds-RNA introduced into acultured cell or the like is degraded into small RNA of about 22nucleotides, and such small RNA hybridizes to mRNA to induce degradationof mRNA (Tuschl, T. et al., Nature Biotech., 2002, vol. 20, pp. 446-448;JP Patent Publication (Kohyo) No. 2003-529374 A (WO 01/75164); and WO02/44321). It has been confirmed by many experiments that degradation ofmRNA is induced by long ds-RNA in the case of Drosophila or nematodes.In the case of animal cells, it has been confirmed that degradation ofmRNA is induced by small ds-RNA of 20 to 30 nucleotides. It is alsosuggested that long ds-RNA disrupts a large number of mRNA molecules ina non-sequence-specific manner (Elbashir, S M. et al., Nature, 2001,vol. 411, pp. 494-498; and WO 00/175164).

A large number of polymerase-III based siRNA expression systems used inorder to effect RNAi in animal cells have been reported (for example,Miyagishi, M. et al., Nature Biotechnol., 2002, vol. 20, pp. 497-500;and Hasuwa, H. et al., FEBS Lett., 2002, vol. 532, pp. 227-230). In thecase of siRNA-based RNAi, however, it is difficult to select a targetsite, the expression at which is to be suppressed. Thus, there is noeffective method of predication at this moment. Since polymerase-IIIpromoters are active in all tissues, it is impossible to causetissue-specific RNAi.

In the case of polymerase II-based RNAi in animal cells, it is pointedout that kinase is activated by ds-RNA upon viral infection or the like,and an interferon signal transduction pathway is activated (Kaufman, RJ. et al., Proc. Natl. Acad. Sci. U.S.A., 1999, vol. 96, pp.11693-11695). When particularly long ds-RNA is expressed in animal cellsduring the production of transgenic animals as described above, it isalso reported that protein synthesis is inhibited in anon-sequence-specific manner and a pathway inducing an interferon isactivated (Elbashir, S. M. et al., Nature, 2001, vol. 411, pp. 494-498).

Accordingly, it is necessary to overcome the problems as mentioned aboveand to efficiently cause RNAi in animal cells.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for simplyand rapidly producing target gene-knockdown animals and a tool therefor.

The present inventors have conducted concentrated studies in order toattain the above object. As a result, we considered that production of atransgenic animal that is capable of expressing ds-RNA for a target genein a specific tissue would enable production of a target gene-knockdownanimal in a simple and rapid manner. Thus, the present inventorsselected the Ski transcription factor as a target gene, prepared avector that would express 540 bp ds-RNA corresponding to the mRNAsequence of the Ski gene, and produced a transgenic mouse into which theresulting expression vector has been introduced. As a result, weconfirmed that the aforementioned transgenic mouse exhibitedabnormalities such as abnormal morphogenesis or hemorrhage during thedevelopmental stage, as with the case of conventional Ski mutant mice.Therefore, the present inventors found that a target gene-knockdownmouse could be produced within a short period of time via utilization ofRNA interference using the expression vector constructed in theaforementioned manner. This has led to the completion of the presentinvention.

More specifically, the present invention relates to a double-strandedRNA (ds-RNA) expression vector that comprises the following sequences(a) to (c):

(a) the following nucleotide sequence (a-1) or (a-2):

-   -   (a-1) a nucleotide sequence encoding all or a part of the target        gene; or    -   (a-2) a nucleotide sequence encoding DNA that hybridizes under        stringent conditions to DNA consisting of a sequence        complementary to the nucleotide sequence (a-1);

(b) a nucleotide sequence complementary to the nucleotide sequence (a)and an inverted repeat thereof; and

(c) a sequence encoding a loop region and connecting the nucleotidesequence (a) to the nucleotide sequence (b),

wherein the above sequences are transcribed into RNA and thereby formingds-RNA having a stem-loop structure.

The ds-RNA expression vector preferably comprises a polymerase IIpromoter or a developmental-stage-specific promoter. An example of suchpromoter is a cytomegalovirus (CMV) early gene promoter.

The ds-RNA expression vector preferably comprises a sequence thatautocatalytically cleaves RNA located upstream of the nucleotidesequences (a) to (c). An example of such sequence is, but is not limitedto, a ribozyme site.

The ds-RNA expression vector preferably comprises a sequence that pausesRNA polymerase located downstream of the nucleotide sequences (a) to(c). An example of such sequence is, but is not limited to, a sequenceof the MAZ domain.

Examples of the nucleotide sequence (c) of the ds-RNA expression vectorinclude those shown in SEQ ID NOs: 2, 5, and 6.

An example of the target gene of the ds-RNA expression vector is adisease-associated gene, and a specific example thereof is the Ski gene.A part of the target gene may be a 540 bp 5′-region of the Ski gene.

The present invention also relates to a target gene-knockdown animal inwhich ds-RNA for the target gene is expressed, preferably the ds-RNA forthe target gene is tissue-specifically expressed.

Examples of the animal include a transgenic animal into which theaforementioned ds-RNA expression vector has been introduced andexpressing ds-RNA for the target gene and a progeny thereof.

When the target gene is a disease-associated gene, for example, theanimal can be an animal model for disease. When the Ski gene is selectedas the target gene, the animal can be an animal model for diseaseexhibiting a disease selected from the group consisting of neural tubeclosure defect, malformation of the iris, and hemorrhage in the head.

An example of the animal is, but is not limited to, a mouse.

The present invention also relates to a method for producing a targetgene-knockdown animal, wherein a ds-RNA expression vector that iscapable of expressing ds-RNA for the target gene is introduced to formds-RNA for the target gene.

In this method, either of the aforementioned ds-RNA expression vectorsis preferably used.

When a disease-associated gene is selected as the target gene, an animalmodel for disease can be produced as the animal. When the Ski gene isselected as the target gene, for example, animal models of diseaseexhibiting a disease selected from the group consisting of neural tubeclosure defect, malformation of the iris, and hemorrhage in the head canbe constructed.

In this method, a preferable example of such an animal is, but is notlimited to, a mouse.

The present invention further relates to an animal cell into which theaforementioned ds-RNA expression vector has been introduced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the structure of the Ski double-stranded (ds)-RNAexpression vector.

FIG. 1B schematically shows a region encoding Ski ds-RNA.

FIG. 2 shows the predicted secondary structures of RNA transcribed froma variety of constructed plasmids.

FIGS. 3A and 3B are photographs showing the results of Western blotanalysis demonstrating a decrease in the Ski protein level in a culturedcell into which the Ski ds-RNA expression vector has been introduced.

FIG. 4 is a photograph showing the results of Northern blot analysisdemonstrating the RNA level that is present in the nucleus and in thecytoplasm of a cell into which various types of Ski ds-RNA expressionvectors have been introduced.

FIG. 5 is a photograph demonstrating that expression of long ds-RNA in anucleus does not affect the phosphorylation of eIF2α.

FIG. 6A is a photograph showing the results of RT-PCR analyzing Ski mRNAdegradation by a Ski ds-RNA expression vector.

FIG. 6B is a photograph showing the results of RT-PCR analyzing Ski mRNAdegradation by a Ski ds-RNA expression vector over time.

FIG. 7A to 7C each independently show the effects of long ds-RNAexpressed from the ds-RNA expression vector on luciferase activity. FIG.7A shows Photinus pyralis luciferase (Pp-luc) activity plotted inarbitrary luminescence units (au). FIG. 7B shows Renilla reniformisluciferase (Rr-luc) activity plotted in arbitrary luminescence units.FIG. 7C shows the results concerning ratios of normalized target tocontrol luciferase.

FIG. 8A is a photograph showing the results of Southern blot analysisdemonstrating the presence of a transgene in a Ski ds-RNA transgenicmouse embryo.

FIG. 8B is a photograph showing the results of Northern blot analysisdemonstrating the presence of the siRNA in a Ski ds-RNA transgenic mouseembryo.

FIG. 9 is a photograph showing neural tube closure defects of a Skids-RNA transgenic mouse.

FIG. 10 is a photograph showing malformation of the iris in an eye of aSki ds-RNA transgenic mouse.

FIG. 11 is a photograph showing hemorrhage in the head of a Ski ds-RNAtransgenic mouse.

FIGS. 12A to 12G are photographs showing abnormalities caused by Ski RNAdeficiency in a Ski ds-RNA transgenic mouse.

FIG. 12A shows hematoxylin-eosin staining of head sections.

FIG. 12B shows Ski mRNA expression in the midbrain and in the mesenchymeas detected by RNA in situ hybridization.

FIG. 12C shows Odc mRNA expression in the midbrain and in the mesenchymeas detected by RNA in situ hybridization.

FIG. 12D shows apoptotic cells assessed by the TUNEL assay on theneuroepithelium of the midbrain and the mesenchyme.

FIG. 12E shows hematoxylin-eosin staining of eyes.

FIG. 12F shows Ski mRNA expression in eyes as detected by in situhybridization.

FIG. 12G shows the results of the TUNEL assay on eyes.

In FIGS. 12A to 12G, “a” represents a wild-type mouse, “b” represents apDECAP-β-gal transgenic mouse, “c” represents a pDECAP-Ski transgenicmouse, and “d” represents a Ski-deficient mouse (Ski^(−/−)). In FIGS.12A and 12E, “MB” represents the midbrain, “II” represents the secondventricle, “III” represents the third ventricle, “O” represents theoptic cup, and “L” represents the lens vesicle.

PREFERRED EMBODIMENTS OF THE INVENTION

The present invention is hereafter described in detail. This patentapplication claims priority from Japanese Patent Application No.2002-314764 filed on Oct. 29, 2002, and encompasses part or all of thecontents as disclosed in the description and/or drawings thereof.

The present invention relates to a target gene-knockdown transgenicanimal via RNA interference and a tool (a double-stranded RNA expressionvector) and a method for simply and rapidly producing such transgenicanimal.

The present inventors considered that selection of an adequate lengthfor ds-RNA, an adequate promoter, and the like is important in order toallow ds-RNA for the target gene to express in a specific tissue whenproduction of a transgenic animal is intended via RNA interference.Accordingly, an experiment was carried out using the transcriptionfactor, Ski, for which the phenotype of the mutant mouse was analyzed indetail. As a result, the present inventors confirmed the expression ofds-RNA in transgenic animals. Since Ski mutation is known to causevarious types of malformation during the fetal stage, it was possible todetermine during the early developmental stage whether the target genewas knocked-down or not based on the expression and the formation ofds-RNA.

Hereafter, the target gene-knockdown animal, the animal model fordisease according to the present invention, and a method for producingsuch animal models are described.

1. RNA Interference

The “RNA interference” refers to the phenomenon described below. Whendouble-stranded RNA (herein also referred to as “ds-RNA”) is present ina cell, endogenous mRNA of a gene that is homologous with the nucleotidesequence of the RNA is degraded, and the gene expression in the cell isthen suppressed in a specific manner. It is also referred to as RNAinterference (RNAi). At first, RNA interference was found in nematodesand then observed in Drosophila, plants, mammalian cells, and the like.In the case of Drosophila, for example, a technique is known in whichhairpin ds-RNA is constructed, the resultant is recognized as ds-RNA inthe cell, and endogenous mRNA is then degraded and disrupted. A desiredgene can be knocked down by setting such desired gene as the geneencoded by such hairpin ds-RNA. However, many of the mechanisms of RNAinterference in a mammal are not yet revealed, RNAi is caused by trialand error at present, and this technique is not yet established as ageneralized technique. When expression of ds-RNA is intended in amammal, RNA interference is not always caused due to position effects orother conditions. Further, when particularly long ds-RNA is allowed toexpress in an animal cell, inhibition of protein synthesis in anon-sequence-specific manner or activation of an interferon-inducingpathway has been reported.

2. Construction of ds-RNA Expression Vector

The ds-RNA expression vector according to the present invention(hereinafter referred to as “the vector of the present invention”) canbe introduced into an animal cell and an animal so that ds-RNA for thetarget gene necessary for RNA interference is expressed and formed. Inthe present invention, “expression” refers to the phenomenon such thatDNA encoding ds-RNA is transcribed into mRNA being capable of forming ads-RNA, in the case of ds-RNA. In general, “expression” refers to thefact that DNA encoding a gene is transcribed into mRNA and mRNA isfurther translated into a protein. In the present invention, the term“expression” refers to both thereof. The term “ds-RNA for the targetgene” refers to ds-RNA that induces RNA interference on the target gene.More specifically, it refers to ds-RNA having a sequence homologous tothat of the target gene. Double-stranded RNA for this target gene isencoded on the vector of the present invention by the followingsequences (a) to (c):

(a) the following nucleotide sequence (a-1) or (a-2):

-   -   (a-1) a nucleotide sequence encoding all or a part of the target        gene; or    -   (a-2) a nucleotide sequence encoding DNA that hybridizes under        stringent conditions to DNA consisting of a sequence        complementary to the nucleotide sequence (a-1);

(b) a nucleotide sequence complementary to the nucleotide sequence (a)and an inverted repeat thereof; and

(c) a sequence encoding a loop region and connecting the nucleotidesequence (a) to the nucleotide sequence (b). Upon the above sequencesare transcribed into RNA, double-stranded RNA (ds-RNA) having astem-loop structure is formed. In the present invention, the term“stem-loop structure” refers to a structure consisting of a stem regionthat complementarily forms double strands and a loop region in a singlestranded loop form that connects these two strands. Such structure isknown in the field of molecular biology.

The nucleotide sequence (a) is a nucleotide sequence that encodes all ora part of the target gene to be knocked down (a-1). The target gene isnot particularly limited as long as it is intended to be knocked downvia RNA interference. Any gene that is deduced to be associated withdisease or other functions, or that is to be studied can be employed asthe target gene. Specific examples thereof include, but are not limitedto, the Ski genes, other transcription factors, and cancer-associatedgenes.

The Ski gene was first identified as an oncogene, and functions thereofas a transcription corepressor was revealed in recent years (Nomura, T.et al., Genes and Development 13: 412-423, 1999). A transcriptioncorepressor is a factor that represses transcription by binding tovarious types of repressors. One species thereof, Ski, plays key rolesin various developmental stages. The Ski genes are isolated from humans,mice, frogs, and flies, the nucleotide sequences thereof are known andregistered at GenBank (Accession Nos: X15218 (human), AF435852 (mouse),X68683 (frog), and NT_(—)033778 (fly)).

In the present invention, the sequence (a) can be designed based on suchpublicly available nucleotide sequence information. Alternatively, aperson skilled in the art can prepare DNA having the sequence (a) via aconventional technique if such DNA can be introduced into the ds-RNAexpression vector without the need to know all the sequences. “A partof” the target gene refers to a sequence of a length that is sufficientfor target gene-knockdown via RNA interference, such as a sequence of500 to 1,000 bp, and preferably a sequence of 500 to 700 bp. When theSki gene is employed, for example, a sequence of approximately 500 to1,000 bp corresponding to the N-terminal region of the Ski protein ispreferable. In the present invention, use of a 540 bp 5′-region (SEQ IDNO: 1) of the Ski gene as the nucleotide sequence (a) is particularlypreferable. SEQ ID NO: 1 is derived from the mouse Ski gene (GenBankAccession No: AF435852) and corresponds to a sequence consisting ofnucleotides 1 to 540 thereof. The vector of the present invention canexpress long ds-RNA of approximately 500 to 1,000 bp and can induce RNAinterference. Thus, a ds-RNA expression vector can be simply and easilyconstructed without the need to select a region (of approximately 20 to30 bp) for specifically degrading endogenous RNA, unlike theconventional case where siRNA (small interfering RNA) is employed.

The nucleotide sequence (a) also includes a nucleotide sequence (a-2)that hybridizes under stringent conditions to DNA consisting of asequence complementary to the nucleotide sequence encoding all or a partof the target gene. Such nucleotide sequence is also capable ofeffectively causing RNA interference if it has a certain degree ofhomology to the target sequence. Under stringent conditions, a specifichybrid is formed but a non-specific hybrid is not formed. Under suchconditions, for example, DNA having high homology (60% or higher andpreferably 80% or higher) with other DNA is capable of hybridizing tothe other DNA. More specifically, a sodium concentration is between 150mM and 900 mM, and preferably between 600 mM and 900 mM, and temperatureis between 60° C. and 68° C., and preferably 65° C., under stringentconditions.

The nucleotide sequence (b) is complementary to the nucleotide sequence(a) and is an inverted repeat thereof. The term “complementary” usedherein does not refer to precise complementarity. It would be sufficientif double strands could be complementarily formed with the nucleotidesequence (a). The “inverted repeat” concerns the direction of thesequence that is inserted into a vector. When the nucleotide sequence(a) is inserted in the form of ATCGAGTC, for example, the nucleotidesequence (b) is inserted in the form of GACTCGAT.

The nucleotide sequence (c) encodes a loop region, and it connects thenucleotide sequence (a) to the nucleotide sequence (b). The term “loopregion” refers to a portion of nucleotide sequence that takes the formof a single strand loop without self-association of nucleotide sequencesin such region based on complementarity or via other means. A techniquefor designing a nucleotide sequence that encodes such a loop region iswell-known in the art. The length of a loop region is approximately 6 bpto 15 bp, and preferably approximately 12 bp. The nucleotide sequences(c) that are preferable in the present invention are shown in SEQ IDNOs: 2, 5, and 6. It should be noted that sequences that can be employedin the present invention are not limited thereto.

As shown in FIG. 1B, the nucleotide sequence (a) is connected to thenucleotide sequence (b) via the nucleotide sequence (c). Upontranscription of these sequences into RNA, accordingly, they formdouble-stranded RNA (ds-RNA) having a stem-loop structure viaself-association of the nucleotide sequences (a) and (b) based oncomplementarity. The resulting ds-RNA induces RNA interference on thetarget gene.

A promoter and/or other control sequence may be operably ligated andinserted into the vector of the present invention so that the sequenceof the ds-RNA region is then transcribed and ds-RNA is formed. Thephrase “operably ligated and inserted into the vector of the presentinvention” refers to a procedure whereby a promoter and/or other controlsequence are ligated and introduced into the cell or the transgenicanimal to which the vector of the present invention is to be introducedso that ds-RNA for the target gene is expressed and formed under thecontrol of the promoter and/or the other control sequence and mRNA ofthe endogenous target gene is disrupted and degraded. Promoters and/orcontrol sequences that can be incorporated into the vector of thepresent invention are not particularly limited. Any promoter and/or acontrol sequence known in the art can be adequately selected inaccordance with various conditions such as the type of animal used andproperties of the target gene to be knocked-down. Examples thereofinclude a constitutive promoter, a tissue-specific promoter, atime-specific promoter, and other regulatory elements. In the presentinvention, a polymerase II promoter is preferably used. This is becausea polymerase II promoter induces tissue-specific gene expression andthus tissue-specific expression of ds-RNA can occur in an organism.

When a Ski-knockdown animal model for disease is prepared, for example,a developmental-stage-specific promoter is preferably incorporated intothe vector of the present invention since Ski-gene-associated diseasesare known to develop during the developmental stage.

In the present invention, the term “developmental-stage-specificpromoter” refers to a promoter that has been demonstrated to drive anexpression specifically during the developmental stage. Such promotersare known in the art. Examples thereof include, but are not limited to,a cytomegalovirus (CMV) early gene promoter, an insulin gene promoter, aLck gene promoter, a CD19 gene promoter, and a Nestin gene promoter.These promoters are known in the art, and a person skilled in the artcan easily obtain the sequence information thereof.

In animal cells, ds-RNA may inhibit various types of mRNA translationupon transportation thereof from the nucleus to the cytoplasm, or it mayactivate ds-RNA-dependent kinase. In order to avoid such problems, asequence that autocatalytically cleaves RNA and/or a sequence thatpauses RNA polymerase may be ligated to the vector of the presentinvention. The term “sequence that autocatalytically cleaves RNA” refersto a sequence that can autocatalytically cleave RNA at the site of suchsequence. More specifically, it refers to a sequence thatautocatalytically cleaves RNA by the ribozyme activity (Huang, Y., Mo.Cell Biol. 16: 1534-1542, 1996). A person skilled in the art can easilydesign the nucleotide sequence of the sequence that autocatalyticallycleaves RNA. An example thereof is, but is not limited to, any ribozymesequence, such as the sequence as shown in SEQ ID NO: 3. Since the capstructure at the 5′ end is critical for transportation of mRNA to thecytoplasm (McKendrick, L. et al., Mol. Cell Biol., 21: 3632-3641, 2001),a sequence that autocatalytically cleaves RNA is inserted immediatelydownstream of the transcription initiation site of ds-RNA for the targetgene, and the cap structure is removed from the ds-RNA synthesized bytranscription. Thus, the aforementioned problem can be avoided.Existence of the sequence that autocatalytically cleaves RNA can resultin autocatalytic cleavage of the transcribed ds-RNA at the site of suchsequence, and the cap structure is then removed.

The term “sequence that pauses RNA polymerase” refers to a sequence thatpauses RNA polymerase on the template DNA to terminate itstranscription. More specifically, it refers to a sequence to which atranscription factor binds (Yonaha, M. et al., Mol. Cell 3: 593-600,1999; Yonaha, M. et al., EMBO J. 19: 3770-3777, 2000). A person skilledin the art can easily design such sequence that pauses RNA polymerase.An example thereof is, but is not limited to, a sequence of atranscription factor, i.e., the MAZ domain. An example of the sequenceof this MAZ domain is shown in SEQ ID NO: 4, although this example isnot exclusive. A poly(A) tail at the 3′ end is important in order totransport mRNA to the cytoplasm (Huang, Y., cited above), and mRNA istransported to the cytoplasm due to the existence of the poly(A) tail.In the present invention, however, it is necessary to retain the ds-RNAformed upon transcription in the nucleus and induce RNA interference.This can be realized by removing the poly(A) tail; however, a poly(A)addition signal is generally essential in order to terminatetranscription. The present inventors considered that the aforementionedproblem could be resolved by replacing the poly(A) addition signal onthe vector with a sequence that terminates transcription (e.g., thesequence of the MAZ domain). Because of the existence of the sequencethat pauses RNA polymerase, transcription of ds-RNA is terminated at thesite of such sequence, and no poly(A) tail is added to the ds-RNA to besynthesized.

In order to insert the aforementioned constituents into an adequatevector, purified DNA is first cleaved with an adequate restrictionenzyme, the cleaved DNA is inserted into the restriction enzyme site ormulti-cloning site of an adequate vector DNA, and the resultant is thenligated to the vector. DNA (the aforementioned constituent) can besynthesized and purified by a technique known in the art.

Examples of vectors that can be used in the present invention includeplasmid DNA, cosmid DNA, bacterial artificial chromosome (BAC) DNA,retrotransposon DNA, yeast artificial chromosome (YAC) DNA, and PIphage-derived artificial chromosome (PAC) DNA. A short pUC plasmid(e.g., a plasmid of approximately 1 kbp to 3 kbp) is particularlypreferable.

The thus obtained vector induces expression and formation of ds-RNA forthe target gene upon introduction thereof into a cell, tissue, and/ororganism, which leads to disruption and degradation of mRNA of theendogenous target gene. In this description, such vector is referred toas a pDECAP vector (Deletion of Cap structure and poly(A)).

The ds-RNA expression vector can preferably suppress tissue-specificexpression of the gene product encoded by a specific endogenous gene.Accordingly, the ds-RNA expression vector of the present invention isuseful for a variety of applications such as analysis of gene functionsand gene therapy.

3. Cell into which ds-RNA Expression Vector has been Introduced

In the present invention, a ds-RNA expression vector that expressesds-RNA for the target gene can be introduced into a cell, therebyallowing ds-RNA for the target gene to express therein. Since mRNA ofthe endogenous target gene is disrupted upon formation of ds-RNA for thetarget gene, expression of the target gene can be suppressed.

Cells into which the ds-RNA expression vectors have been introduced arenot particularly limited. Animal cells, such as cells of humans, mice,cattle, sheep, goats, pigs, rabbits, and rats, are preferable.

The ds-RNA expression vector can be introduced into cells via any meanswithout particular limitation. For example, a method involving the useof calcium ions and electroporation can be employed.

The thus prepared cells suppress the expression of the target gene.Therefore, they are useful for analyzing gene functions and ex vivosuppression of the target gene expression.

4. Production of Transgenic Animal (Target Gene-Knockdown Animal)

The target gene-knockdown animal (transgenic animal) of the presentinvention has ds-RNA for the target gene, and mRNA of the endogenoustarget gene is disrupted due to the formation of ds-RNA for the targetgene. This transgenic animal can be generated by introducing the ds-RNAexpression vector into an animal cell and then allowing the animal cellto grow. In the thus generated transgenic animal, target gene knockdownis realized via disruption and degradation of mRNA of the endogenoustarget gene due to the presence of ds-RNA. In the present invention, theterm “endogenous” refers to the natural presence of a gene in the celland/or the organism to which the vector of the present invention is tobe introduced. It also refers to the presence therein of a gene sincebefore the vector was introduced. The term “knockdown” used hereinrefers to reduced expression of a gene product (e.g., a protein).Animals to which the ds-RNA expression vector of the present inventionis to be introduced are not particularly limited as long as they aremammals. Examples thereof include mice, cattle, sheep, goats, pigs,rabbits, and rats. In the present invention, mice, rats, and the likeare preferable since they are easy to handle as animal models ofdisease.

The vector of the present invention is preferably introduced into ananimal cell by injection into the fertilized egg of the target animal.As a result, the sequence encoding the ds-RNA for the target gene and acontrol sequence are incorporated into the fertilized egg cell, and thesequence encoding the ds-RNA and a control sequence are copied in cellsduring division of the egg.

Genes can be introduced into animal cells using techniques such as themethod of introduction into ES cells and the method of introducing acell nucleus introduced into the culture cells via nucleartransplantation into fertilized eggs, in addition to microinjection intofertilized eggs. DNA of the vector of the present invention can beintroduced into ES cells or other culture cells by electroporation,lipofection, or other means and can be subjected to positive selectionwith neomycin, promycin, or the like. Thus, the cells of interest may beobtained. ES cells are injected into nascent embryos or 8-cell-stageembryos using capillaries or the like. Nuclear transplantation iscarried out by injecting the cells into which DNA has been introducedinto the fertilized eggs from which the nucleus has been removed, andcell fusion is carried out by electrical stimulation.

Thereafter, nascent embryos or 8-cell-stage embryos are directlytransplanted into the ovarian duct of a foster mother. Alternatively,nascent embryos or 8-cell-stage embryos are cultured for a day andembryos that have developed to blastocysts are then transplanted intothe uterus of a foster mother. The foster mother is raised and made togive birth to offspring to obtain a transgenic animal (chimeric animal),which is an offspring into which the target ds-RNA has been introduced.In order to confirm the expression of a desired ds-RNA in the animal, apart of the animal body (for example, a caudal tip) is collected, DNA inthe somatic cell is extracted, and the presence of the introducedsequence is confirmed by PCR or Southern blotting. The animal, once thepresence of the sequence introduced therein has been confirmed, isdetermined to be the first generation, and 50% of the ds-RNA is thengenetically transmitted to its offspring (F1). More specifically, matingbetween a transgenic animal and a normal animal can produce aheterozygous animal (F1), and mating between heterozygotes can produce ahomozygous animal (F2). Further, the offspring of the targetgene-knockdown animal can be obtained by repeating mating betweenanimals of the same species.

Thus, a transgenic animal with specific target gene knockdown is usefulas an animal model for disease or as an animal for genetic analysis asdescribed below.

5. Animal Model for Disease

If the target gene of the transgenic animal produced in the mannerdescribed above is knocked down (mutated), this transgenic animal can beutilized as an animal model exhibiting several types of diseases (ananimal model for disease). For example, a transgenic Ski-knockdownanimal may exhibit neural tube closure defects, malformation of the irisin the eye, or hemorrhage in the head. Hemorrhage in the head was firstfound by the present inventors. Accordingly, the animal model fordisease according to the present invention is useful for research ofsuch diseases, development of therapy techniques for such diseases, andother purposes.

EXAMPLES

The present invention is hereafter described in greater detail withreference to the examples, although the technical scope of the presentinvention is not limited thereto.

Example 1 Construction of Ski Double-Stranded RNA (ds-RNA) ExpressionVector

A plasmid that allows the expression of hairpin RNA (i.e., RNAcomprising a self-complementary stem loop) was constructed as follows:as shown in FIG. 1A, a 540 bp 5′-region (SEQ ID NO: 1) in coding regionof the Ski gene was inserted into the pDECAP plasmid (shown below), anda sequence complementary to the aforementioned 40-bp sequence wasinserted downstream thereof as an inverted repeat separated by a 12-bpspacer (the loop region) as shown in SEQ ID NO: 2. The resultant wasdesignated as pDECAP-Ski.

Sequence of the spacer: 5′-GGTGCGCATATG-3′ (SEQ ID NO: 2)

pDECAP comprises pcDNA3 (Invitrogen) having a CMV promoter, a ribozymecassette cutting off the cap structure at the 5′ end of RNA transcribedas a sequence that autocatalytically cleaves RNA (SEQ ID NO: 3; Huang,Y. and Carmichael, G C, 1996, Mol. Cell Biol. 16: 1534-1542), and a MAZdomain as a sequence for pausing RNA polymerase II transcription (SEQ IDNO: 4; Yonaha, M. and Proudfoot, N J., 1999, Mol. Cell 3: 593-600). Thisconstruct does not have any poly(A) addition signal.

mRNA transcribed from this expression vector forms a stem-loop structureat the 12-bp spacer portion thereof and becomes ds-RNA having thesequence of Ski mRNA.

In the same manner as described above, the first 518 bp of the fireflyluciferase-gene-encoding region or the sequence consisting ofnucleotides 443-944 of the β-galactosidase gene are separated by a 12-bpspacer (SEQ ID NO: 5 or 6, respectively) from a sequence complementarythereto as an inverted repeat thereof. Thus, a fireflyluciferase-targeted ds-RNA expression vector (pDECAP-Luc) and aβ-galactosidase-targeted ds-RNA expression vector (pDECAP-β-gal) wereconstructed.

The pDECAP plasmids containing the inverted repeats, thus constructed,were amplified in the E. coli Sure 2 strain (Stratagene), which allowsthe accurate replication of DNA containing inverted repeats, andpurified using the EndoFree Plasmid Maxi Kit (Qiagen).

The pDECAP-LucS plasmid expressing sense RNA of the firefly luciferasegene, the pDECAP-LucAS plasmid expressing antisense RNA of the fireflyluciferase gene, a common pCMV-LucS plasmid expressing RNA of thefirefly luciferase gene containing the 5′-cap structure and a poly(A)tail, and the pCMV-gali plasmid containing a 5′-cap structure and apoly(A) tail and expressing ds-RNA for β-galactosidase were constructed.

The predicted secondary structures of RNAs transcribed from the plasmidsthus constructed are shown in FIG. 2. pDECAP-Ski, pDECAP-Luc, andpDECAP-β-gal encode a 540- to 502-bp hairpin ds-RNA for Ski, fireflyluciferase, and β-galactosidase, respectively, which lack the 5′-capstructure and a poly(A) tail. Sense and antisense single-stranded RNA ofthe luciferase gene were transcribed from pDECAP-LucS and pDECAP-LucAS,respectively. pCMV-gali encodes the same hairpin ds-RNA as pDECAP-β-gal,except that it contains the 5′-cap structure, a splicing intron, and apoly(A) tail.

Example 2 Reduced Ski Expression Level Caused by Ski ds-RNA ExpressionVector

In this example, the effect of the Ski ds-RNA expression vector on theSki protein level in cultured cells was examined.

(1) Southern Blotting

293T cells from human kidney were cultured in DMEM (Invitrogen)containing 10% FBS and an antibiotic (penicillin/streptomycin;Invitrogen). The pDECAP-Ski plasmid prepared in Example 1 and 0.05 μg ofTK-Luc (a firefly luciferase expression vector; Promega) as the internalcontrol were transfected into 4×10⁵ 293T cells using Lipofectamine(GibcoBRL). Cells were lysed in Passive Lysis Buffer (Promega) 42 hourslater, and aliquots of the cell extracts were subjected to luciferaseassay (Promega) to evaluate the transfection efficiency.

The remaining cell pellets were resuspended in a RIPA lysis buffer (1%Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.01M sodiumphosphate (pH 7.2), and 1% trasylol), and the resultant was adjusted inaccordance with the luciferase assay results. A mixture of anti-Skimonoclonal antibodies 1-1, 9-1, 11-1, and 16-1 (Nomura et al., 1999,supra) was used for immunoblotting (Western blotting) in accordance withthe method of Shinagawa et al. (Shinagawa, T, et al., 2000, EMBO J., 19:2280-2291).

A band of approximately 90 kD for Ski protein was detected by Westernblotting. A mouse Ski expression vector (pact-ski; Nomura, T. et al.,Genes & Development 13: 412-423, 1999) and various amounts of Ski ds-RNAexpression vectors (pDECAP-Ski) were introduced to perform Westernblotting in the same manner. As a result, the Ski protein level wasfound to decrease depending on the amount of ds-RNA (FIG. 3A).

As a negative control, the pDECAP-β-gal plasmid prepared in Example 1was also introduced into the 293T cell together with the mouse Skiexpression vector (pact-Ski) to analyze the Ski expression level. Inthis case, the Ski protein level was not decreased (FIG. 3A).Accordingly, Ski ds-RNA was found to decrease the Ski expression levelspecifically in cultured animal cells.

The pDECAP-Ski and pDECAP-β-gal plasmids were transfected into the 293Tcells, and endogenous Ski and Sno proteins were detected byimmunoblotting. When the pDECAP-Ski vector was independently transfectedinto the 293T cell, the level of endogenous Ski protein decreased (FIG.3B). The level of the endogenous Sno protein having approximately 60%homology to the target sequence of pDECAP-Ski did not change.

(2) Northern Blotting 293T cells were transfected with 0.8 μg of varioustypes of expression vectors shown in FIG. 2 (pDECAP-Ski, pDECAP-Luc,pDECAP-β-gal, pDECAP-LucS, pDECAP-LucAS, pCMV-LucS, or pCMV-gali).Cytoplasmic and nuclear RNAs were extracted therefrom 48 hours later.The subcellular distribution of RNAs was examined by Northern blottingusing a digoxigenin (DIG)-labeled LucS DNA probe.

The results are shown in FIG. 4. The upper photograph in FIG. 4 showsthe results of Northern blotting, and the lower photograph shows thecontrol in order to demonstrate that the RNA levels are the same in alllanes. While most of the luciferase sense RNAs transcribed frompDECAP-LucS were in the nucleus, the luciferase sense RNA transcribedfrom pCMV-LucS was in the cytoplasm. The size of the luciferasetranscripts from pDECAP-LucS corresponds to that of a plasmid. Thissuggests that the long RNAs were transcribed by a read-through mechanismdue to a lack of the poly(A) addition signal. The long RNA transcribedfrom pDECAP-Luc was detected neither in the cytoplasm nor in the nucleusby Northern blotting. This suggests that it was immediately digestedinto small RNAs.

(3) Interferon Response

To investigate whether Ski mRNA expressed from pDECAP-Ski induces aninterferon response, the phosphorylation status of eIF2α, which is asubstrate of the ds-RNA-activated PKR protein kinase, was examined. 293Tcells were cotransfected with pDECAP empty plasmid or pDECAP-Ski plasmid(0.6 μg) and pCMV-β-gal plasmid (0.3 μg) as an internal control. Thecells were harvested 48 hours later and subjected to immunoblot analysisusing a phospho-specific antibody against eIF2α phosphorylated on Ser 51(eIF2α-P). The same membrane was reprobed with antibodies against totaleIF2α and β-galactosidase. The lysate of 293T cells treated withpoly(I)•poly(C) (100 μg/ml) for 24 hours was used as a positive control.

The results are shown in FIG. 5. The levels of phosphorylated eIF2α werenot enhanced by transfection of 293T cells with pDECAP-Ski, whereas thephosphorylation of eIF2α was stimulated in the control cells treatedwith poly(I)•poly(C) (FIG. 5). This indicates that interferon responsewas not induced in the cells into which pDECAP-Ski had been introduced.

(4) RT-PCR Assay

To determine the level of endogenous Ski mRNA, the reverse transcriptasePCR (RT-PCR) was carried out. More specifically, 1.5 μg each of pDECAPempty plasmid, pDECAP-Ski plasmid, and pDECAP-β-gal plasmid weretransfected into 293T cells together with enhanced green fluorescentprotein (EGFP) expression plasmid (pMSCV-ir-EGFP). EGFP-positive cellswere collected by sorting and then treated with actinomycin D. Total RNAwas isolated at various time points and the level of Ski mRNA wasanalyzed by RT-PCR. The RT-PCR products were separated on 2% agarosegels and visualized by ethidium bromide staining.

The results are shown in FIG. 6A and FIG. 6B. FIG. 6A shows the resultsof RT-PCR demonstrating the degradation of Ski mRNA by the Ski ds-RNAexpression vector. FIG. 6B shows the results of RT-PCR that analyzed thedegradation of Ski mRNA by the Ski ds-RNA expression vector over time.

RT-PCR demonstrated that the levels of endogenous Ski mRNA were alsoreduced by transfection with pDECAP-Ski (FIG. 6B). To confirm that thedecreased Ski mRNA levels were due to the degradation of Ski mRNA, SkimRNA levels at various time points were measured after treatment of thetransfected cells with an inhibitor of transcription, i.e., actinomycinD (FIG. 6B). In the cells transfected with the pDECAP-Ski plasmid, thehalf-life of endogenous Ski mRNA was 0.9 hours, whereas it was 2.7 and3.1 hours in cells transfected with the pDECAP plasmid expressingβ-galactosidase ds-RNA (pDECAP-β-gal) and the empty plasmid,respectively. This indicates that Ski ds-RNA expressed from the pDECAPvector stimulated the degradation of Ski mRNA.

Example 3 Luciferase Assay of Mouse Embryonic Fibroblast Cells

Mouse embryonic fibroblast cells were cultured in Dulbecco's ModifiedEagle medium (DMEM) containing 10% fetal bovine serum (FBS) andantibiotics. Cells (0.5×10⁵ cells) were transferred to 12-well plates (1ml per well) and transfected with mixtures of the firefly luciferase(Pp-luc) expression plasmids (0.1 μg, TK-βRex2-Luc, containing thethymidine kinase promoter and β-retinoic acid receptor-binding sites),the plasmids constructed in Example 1 (0.4 μg, pDECAP-ski, pDECAP-Luc,pDECAP-β-gal, pCMV-gali, pDECAP-LucS, and pDECAP-LucAS), and the Renillareniformis luciferase (Rr-luc) expression plasmid (0.02 μg, pRL-CMV)using Lipofectamine (Invitrogen) according to the manufacturer'sinstructions. All-trans retinoic acid (1 μM) was added thereto 24 hourslater to induce expression of the firefly luciferase gene (Pp-luc) andcells were cultured for an additional 36 hours. Luciferase expressionwas subsequently monitored with the dual luciferase assay (Promega).

The results are shown in FIG. 7A to FIG. 7C. FIG. 7A shows fireflyluciferase (Pp-luc) activity plotted in arbitrary luminescence units(a.u.). FIG. 7B shows Renilla reniformis luciferase (Rr-luc) activityplotted in arbitrary luminescence units. FIG. 7C shows ratios of thenormalized target to the control luciferase.

Firefly luciferase (Pp-luc) levels were reduced about fivefold oncotransfection with the pDECAP plasmid (pDECAP-Luc) expressing fireflyluciferase ds-RNA (FIG. 7A). In contrast, Renilla luciferase (Rr-luc)activity was not affected (FIG. 7B). Cotransfection of the pDECAPplasmid (pDECAP-LucS) expressing sense RNA for the same region of thefirefly luciferase gene did not affect the firefly luciferase activity.In contrast, cotransfection of the pDECAP plasmid (pDECAP-LusAS)expressing antisense RNA for the same region had a slight effect (abouta 25% reduction). This indicates that the structure of the ds-RNA iscritical for effectively inhibiting firefly luciferase activity.Further, coexpression of 502 bp ds-RNA for the β-galactosidase gene fromthe pDECAP plasmid did not affect the firefly or Renilla reniformisluciferase activity. In the case of coexpression of ds-RNA forβ-galactosidase containing a 5′-cap structure, splicing sites, and apoly(A) tail from another plasmid (pCMV-gali, which had the CMV promoterbut lacked both ribozyme and MAZ sites), the activities of bothluciferases were decreased. This indicates that the removal of the capstructure and the poly(A) is required to suppress nonspecific inhibitionof translation.

Example 4 Generation of Transgenic Mouse into which Ski ds-RNAExpression Vector has Been Introduced

(1) Southern Blotting

A 2.2 kb BglII-BamHI fragment in pDECAP-Ski or pDECAP-gali was cleavedout of the background sequence, purified, and then injected into a mousefertilized egg. The mouse fertilized egg was derived from mating betweena male C57BL/6N mouse and a female C57BL/6N or (C57BL/6JxDBA) F1 mouse(obtained from CLEA). To examine the presence of the transgenes and thecopy number thereof, the embryo was analyzed by Southern blot analysisutilizing the first 540 bp fragment of the Ski-coding region as a probe.Genomic DNA used for Southern blot analysis was extracted from theextraembryonic tissue. The results are shown in FIG. 8A. In FIG. 8A, theleft lane shows the results of analyzing the pDECAP-Ski transgenic mouseand the right lane shows those for the wild-type mouse (WT). Thisdemonstrates that the transgenic mouse actually had the Ski ds-RNAexpression vector. The transgene copy number was deduced to be 2 to 12.

(2) Northern Blotting

To examine whether the short RNA was generated in the transgenic mouseembryo, Northern blotting was carried out using the Ski sense-strandprobe.

Nuclear RNA (5 μg) was extracted from the head of a transgenic mouseembryo and then analyzed by Northern blotting. The membrane was probedwith a DIG-labeled sense Ski probe corresponding to the target sequence.FIG. 8B (lower part) shows 5S rRNA visualized by staining with methyleneblue on the membrane. The results indicate that the small RNA of about21 or 22 nucleotides was detected in the pDECAP-Ski transgenic mouseembryo but was not detected in the wild-type embryo (FIG. 8B).

(3) Real Time RT-PCR

To analyze the mouse embryos, RNA was isolated from the limb bud of anembryo using ISOGEN (Nippon Gene Co., Ltd.). Expression of transgeneswas assayed via real-time RT-PCR using the primer pair shown below andthe dual-labeled flourescent probe at the 3′ end of the ribozymecassette having the following sequence. (Primer pair)CCGCCTGATGAGTCCGTGAG (SEQ ID NO:7) ATCGAAGTCATGGTGGCTA (SEQ ID NO:8)(Probe) GACGAAACATGCATAGGC (SEQ ID NO:9)

The results of RT-PCR are shown in Table 1. In Table 1, the expressionlevels of the transgenes are represented by relative values when theembryo No. 257 is determined to be 1.00. TABLE 1 Expression levelGenotype Embryo (No.) of transgene Neural tube defects skii 256 0.015 −257 1.00 + 259 <0.002 − 262 4.58 + gali 233 9.15 − 268 <0.002 −

Example 5 Murine Model of Disease

(1) Neural Tube Defects

It has already been reported that Ski mutant mice have neural tubeclosure defects (Berk, M. et al, Genes Dev. 11: 2029-2039, 1997). Thepresent inventors analyzed the Ski ds-RNA-expressing transgenic mice atE10.5 and confirmed that such transgenic mice exhibited similar neuraltube closure defects (FIG. 9). As indicated by an arrow in FIG. 9, theneural tubes of the transgenic (TG) mice were not closed. Among 13transgenic embryos, 5 (38.5%) exhibited neural tube closure defects (seeTable 1). The β-galactosidase ds-RNA-expressing transgenic mice (9 mice)prepared as negative controls did not exhibit neural tube closuredefects.

(2) Defects in Eye Formation

It has already been reported that Ski mutant mice exhibit malformationsof the irises in their eyes (Colmenares, C. et al., Nature Genet. 30:106-109, 2002). The present inventors analyzed the Ski ds-RNA-expressingtransgenic mice at E10.5 and confirmed that such transgenic micedeveloped malformations of the irises in their eyes (FIG. 10). Asindicated by an arrow in FIG. 10, transgenic (TG) mice lack part of theirises in their eyes. Each of two transgenic embryos (100%) exhibitedmalformations of the irises in the eyes. The β-galactosidaseds-RNA-expressing transgenic mice (9 mice) prepared as negative controlsdid not exhibit malformations of the irises in the eyes.

(3) Hemorrhage in the Head

The present inventors observed that the Ski ds-RNA-expressing transgenicmice developed hemorrhages in their heads (FIG. 11). In FIG. 11, thetransgenic (TG) mice and the Ski mutant mice (ski^(−/−)) exhibithemorrhages in their heads. This demonstrates that the Ski mutant micecan be employed as murine models for hemorrhage in the head.

Based on the results shown in (1) to (3) above, the Ski ds-RNAtransgenic mice exhibit defects and hemorrhage in the head, as with theSki mutant mice. In general, the effects of transgenes are observed in20% to 80% of transgenic mice individuals. This is because the transgenemay not be expressed due to the position of the gene integrated(position effects). Neural tube closure defects were observed in 38.5%of the Ski ds-RNA transgenic mice constructed by the present inventors.This rate is reasonable based on past reports concerning transgenicmice.

Example 6 Introduction of ds-RNA and Frequency of Phenotype Expression

Transgenic mouse embryos containing the β-galactosidase expressionvector driven by the CMV promoter (pCMV-β-gal) were prepared in order toexamine whether or not the CMV promoter exhibits activity in variousregions of mouse embryos.

In two of six embryos of pCMV-β-gal transgenic mice, the β-galactosidasewas expressed in almost all tissues, including the neuroepithelialcells, at E9-E12. This frequency (33.3%, 2/6) is similar to that of theembryos of pDECAP-Ski transgenic mice exhibiting abnormalities (Example5) and is within the average frequency (10% to 40%) of transgenic miceshowing the specific phenotype caused by the injected DNA.β-galactosidase expression was frequently observed in various tissues,including the eyes, of the embryos of pCMV-β-gal transgenic mice at E15.This is consistent with the case where β-galactosidase expression wasfrequently observed in embryos of pDECAP-Ski transgenic mice exhibitingeye defects at E14-E16.

As control experiments, transgenic mice were prepared by injecting afragment containing the expression unit of 502-nt β-galactosidase ds-RNAprepared from the pDECAP-β-gal. No abnormality was observed in any ofthe nine mouse embryos analyzed (Table 2). This indicates that theabnormalities observed in the pDECAP-Ski transgenic mice werespecifically caused by Ski ds-RNA, but were not caused by long ds-RNA ina non-specific manner. Although the typical abnormalities ofSki-deficient mice such as neural tube defects and eye defects wereobserved in pDECAP-Ski transgenic mice, other defects such as postaxialpolydactyly of Ski-deficient mice were not observed therein. This may bebecause the CMV promoter was not sufficiently active in some tissues.TABLE 2 Number of embryos exhibiting phenotypes/number of Stage ofanalyzed Number of transgenic embryos analyzed DNA injected embryosembryos obtained Neural tube defect Eye defect β-gal expressionpDECAP-Ski E9-E12 13   5/13 (38.5%)  2/2 (100%)^(a) ND E14-E16 E9-E11 20/2 (0%) 2/2 (100%) ND pDECAP-β-gal E9-E12 9 0/9 (0%) ND ND pCMV-β-galE15 6 0/6 (0%) ND 2/6 (33.3%) 1 0/1 (0%) 0/1 (0%)  1/1 (100%) ^(a)Ski expression in lens vesicle of E10.5 embryos(ND): Not determined

Example 7 Histological Analysis of Transgenic Mice

Hematoxylin-eosin staining of tissue, in situ hybridization using RNA,and Tunel assay were performed in order to examine the influences ofendogenous Ski gene expression and RNAi on transgenic mice.

Hematoxylin-eosin staining was performed by placing samples on glassslides and immersing the glass slides in each solution in the followingmanner:

1) a hematoxylin solution at room temperature for 3 minutes;

2) rinsing under running water;

3) 1% eosin at room temperature for 7 minutes;

4) 50% ethanol at room temperature for 2 seconds 7 times;

5) 70% ethanol at room temperature for 2 seconds 7 times;

6) 90% ethanol at room temperature for 2 seconds 7 times;

7) 95% ethanol at room temperature for 2 seconds 7 times;

8) 100% ethanol at room temperature for 15 minutes;

9) 100% ethanol at room temperature for 15 minutes;

10) 100% ethanol at room temperature for 15 minutes;

11) xylene at room temperature for 15 minutes;

12) xylene at room temperature for 15 minutes;

13) xylene at room temperature for 15 minutes; after which

14) xylene was evaporated to dryness, mounting medium was mounted onglass slides, and coverslips were placed thereon.

In situ hybridization was performed in accordance with the method ofButler, K. et al. (Methods, vol. 23, pp. 303-312, 2001). For detectionof Ski mRNA, the 3′-half of the Ski gene was used as the probe. Morespecifically, a plasmid spanning nucleotides 754-2223 of the mouse c-SkicDNA was linearized with BamHI and transcribed with T7 RNA polymerase toprepare the c-Ski 3′ probe to be used. The ODC probe for Odc genedetection has been already reported by Nomura, T. et al. (Genes & Dev.,1999, vol. 13, pp. 412-423).

The TUNEL assay was employed to study apoptosis using a commerciallyavailable apoptosis kit (in situ cell death detection kit, Roche)according to the manufacturer's instructions.

The results of the above experiments are shown in FIGS. 12A to 12G InFIGS. 12A to 12G, “a” represents a wild-type mouse, “b” represents apDECAP-β-gal transgenic mouse, “c” represents a pDECAP-Ski transgenicmouse, and “d” represents a Ski-deficient mouse (Ski^(−/−)). FIG. 12Ashows hematoxylin-eosin staining of head sections. FIG. 12B shows SkimRNA expression in midbrains and mesenchymes as detected by RNA in situhybridization. FIG. 12C shows Odc mRNA expression in midbrains andmesenchymes as detected by RNA in situ hybridization. FIG. 12D showsapoptotic cells assessed by the TUNEL assay on the neuroepitheliums ofmidbrains and mesenchymes. FIG. 12E shows hematoxylin-eosin staining ofeyes. FIG. 12F shows Ski mRNA expression in eyes detected by in situhybridization. FIG. 12G shows the results of the TUNEL assay on eyes. InFIGS. 12A and 12E, “MB” represents the midbrain, “II” represents thesecond ventricle, “III” represents the third ventricle, “O” representsthe optic cup, and “L” represents the lens vesicle.

In the neural epithelial cells of the pDECAP-Ski transgenic mice and theSki-deficient mice, which were generated by homologous recombination inES cells, Ski mRNA was not detected (FIG. 12B: c and d). In contrast,Ski mRNA was expressed in the neural epithelial cells of pDECAP-β-galds-RNA transgenic mice and wild-type mice (FIG. 12B: a and b). Theseresults were confirmed by RT-PCR (data not shown).

The neural tube closure defects of Ski-deficient mice were caused by theectopic expression of the ornithine decarboxylase (Odc) gene followed byapoptosis in neural epithelial cells. This is because Ski is requiredfor Mad-mediated transcriptional repression of the Odc gene (Nomura, T.et al., Genes & Dev., 1999, vol. 13, pp. 412-423). Similar ectopicexpression of the Odc gene was observed in neural epithelial cells ofembryos of pDECAP-Ski transgenic mice (FIG. 12C: c). Furthermore,ectopic apoptosis was observed in regions where the levels of Odcexpression were high (FIG. 12D: c and d). These results indicate thatthe neural tube closure defects observed in pDECAP-Ski transgenic miceare caused by the same mechanism that operates in Ski-deficient micegenerated by homologous recombination in ES cells.

During eye development, different retinal cell types, including retinalganglion cells, cone photoreceptors, amacrine cells, and rodphotoreceptors, are generated from the retinal progenitor cells in afixed chronological sequence (Young, R. W., Anat. Rec., 1985, vol. 212,pp. 199-205). In embryos of wile-type mice, Ski mRNA was detected in thelens vesicle, optic cup, and surface epithelium at E10.5 (FIG. 12F: a).In embryos of pDECAP-Ski transgenic mice and Ski-deficient mice, SkimRNA levels in these regions were dramatically reduced (FIG. 12F: c andd). Ectopic apoptosis was observed in the lens vesicle and the surfaceepithelium of pDECAP-Ski transgenic mice and Ski-deficient mice, whichwas consistent with the above observations (FIG. 12G: c and d). However,ectopic apoptosis was not observed in embryos of wild-type mice (FIG.12G: a). Ectopic apoptosis of the optic cup was observed in theSki-deficient mouse embryos but was not apparent in embryos ofpDECAP-Ski transgenic mice. This suggests that low levels of Ski mRNAmay remain in this region.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention provides a target gene-knockdown transgenic animaland a ds-RNA expression vector therefor. The transgenic animal accordingto the present invention can be easily and rapidly produced with the useof a ds-RNA expression vector that induces RNA interference. The Skiknockdown transgenic animal according to the present invention can beutilized as, for example, an animal model for exhibiting several typesof diseases. Further, the ds-RNA expression vector according to thepresent invention is constructed to be capable of efficiently inducingRNA interference. Thus, it can express ds-RNA for the target gene andsuppress the expression of the target gene without being affected byposition effects.

Free Text of Sequence Listing

SEQ ID NOs: 2 to 9: synthetic oligonucleotides

1. A double-stranded RNA (ds-RNA) expression vector that comprises thefollowing sequences (a) to (c): (a) the following nucleotide sequence(a-1) or (a-2): (a-1) a nucleotide sequence encoding all or a part ofthe target gene; or (a-2) a nucleotide sequence encoding DNA thathybridizes under stringent conditions to DNA consisting of a sequencecomplementary to the nucleotide sequence (a-1); (b) a nucleotidesequence complementary to the nucleotide sequence (a) and an invertedrepeat thereof; and (c) a sequence encoding a loop region and connectingthe nucleotide sequence (a) to the nucleotide sequence (b), wherein thesequences are transcribed into RNA and thereby forming ds-RNA having astem-loop structure.
 2. The ds-RNA expression vector according to claim1, which further comprises a polymerase II promoter.
 3. The ds-RNAexpression vector according to claim 1, which further comprises adevelopmental-stage-specific promoter.
 4. The ds-RNA expression vectoraccording to claim 2, wherein the polymerase II promoter ordevelopmental-stage-specific promoter is a cytomegalovirus (CMV) earlygene promoter.
 5. The ds-RNA expression vector according to claim 1,which further comprises a sequence that autocatalytically cleaves RNAlocated upstream of the nucleotide sequences (a) to (c).
 6. The ds-RNAexpression vector according to claim 5, wherein the sequence thatautocatalytically cleaves RNA is a ribozyme site.
 7. The ds-RNAexpression vector according to claim 1, which further comprises asequence that pauses RNA polymerase located downstream of the nucleotidesequences (a) to (c).
 8. The ds-RNA expression vector according to claim7, wherein the sequence that pauses RNA polymerase is a sequence of theMAZ domain.
 9. The ds-RNA expression vector according to claim 1,wherein the nucleotide sequence (c) is as shown in SEQ ID NO: 2, 5, or6.
 10. The ds-RNA expression vector according claim 1, wherein thetarget gene is a disease-associated gene.
 11. The ds-RNA expressionvector according to claim 1, wherein the target gene is the Ski gene.12. The ds-RNA expression vector according to claim 11, wherein a partof the target gene is a 540 bp 5′-region of the Ski gene.
 13. A targetgene-knockdown animal, in which a ds-RNA for the target gene isexpressed.
 14. The animal according to claim 13, in which the ds-RNA forthe target gene is tissue-specifically expressed.
 15. The animalaccording to claim 13, which is a transgenic animal having a ds-RNAexpression vector introduced therein and expressing ds-RNA for thetarget gene, or progeny thereof.
 16. The animal according to claim 15,wherein the ds-RNA expression vector comprises the following sequences(a) to (c): (a) the following nucleotide sequence (a-1) or (a-2): (a-1)a nucleotide sequence encoding all or a part of the target gene; or(a-2) a nucleotide sequence encoding DNA that hybridizes under stringentconditions to DNA consisting of a sequence complementary to thenucleotide sequence (a-1); (b) a nucleotide sequence complementary tothe nucleotide sequence (a) and an inverted repeat thereof; and (c) asequence encoding a loop region and connecting the nucleotide sequence(a) to the nucleotide sequence (b), wherein the sequences aretranscribed into RNA and thereby forming ds-RNA having a stem-loopstructure.
 17. The animal according to claim 13, wherein the target geneis a disease-associated gene, and the animal is an animal model fordisease.
 18. The animal according to claim 17, wherein the target geneis the Ski gene, and the disease is selected from the group consistingof neural tube closure defect, malformation of the iris, and hemorrhagein the head.
 19. The animal according to claim 13, wherein the animal isa mouse.
 20. A method for producing a target gene-knockdown animal,wherein a ds-RNA expression vector capable of expressing ds-RNA for thetarget gene is introduced to form ds-RNA for the target gene.
 21. Themethod according to claim 20, wherein the ds-RNA expression vectorcomprises the following sequences (a) to (c): (a) the followingnucleotide sequence (a-1) or (a-2): (a-1) a nucleotide sequence encodingall or a part of the target gene; or (a-2) a nucleotide sequenceencoding DNA that hybridizes under stringent conditions to DNAconsisting of a sequence complementary to the nucleotide sequence (a-1);(b) a nucleotide sequence complementary to the nucleotide sequence (a)and an inverted repeat thereof; and (c) a sequence encoding a loopregion and connecting the nucleotide sequence (a) to the nucleotidesequence (b), wherein the sequences are transcribed into RNA and therebyforming ds-RNA having a stem-loop structure.
 22. The method according toclaim 20, wherein the target gene is a disease-associated gene, and theanimal is an animal model for disease.
 23. The method according to claim22, wherein the target gene is the Ski gene, and the disease is selectedfrom the group consisting of neural tube closure defect, malformation ofthe iris, and hemorrhage in the head.
 24. The method according to claim20, wherein the animal is a mouse.
 25. An animal cell having the ds-RNAexpression vectors according to claim 1 introduced therein.